Step 0: Generate N2 and CB4856

males
place 10 L4s on a fresh
plate

Incubate 5hrs at
30C

Incubate at 20C for three

days

Hunt for
males!

This is a
male
 Step 0: Generate N2 and CB4856
males
place 10 L4s on a fresh
plate

Incubate 5hrs at
30C

Incubate at 20C for three

days

Hunt for
males!

This is a
male
 Step 0: Generate N2 and CB4856
males

Transfer males to plate that has five hermaphrodite L4s

already on it

If you can not find 10 males to transfer, remove L4

hermaphrodites to maintain
2:1 male:hermaphrodite ratio!

This will generate a male stock to use for setting up

crosses
 Step 1: Cross NIL to parental
genotype

5
x1
1 0
0

10 males and 5 hermaphrodites per

plate
10 replicate plate crosses
Step 2: Transfer single hermaphrodites to new plates
and allow to self

Pare Parental Recombina Parental

nt Genotype nt Genotype

When progeny have been generated, genotype the

parental hermaphrodite to verify it was a cross
progeny by PCR
Step 2: Transfer single hermaphrodites to new plates
and allow to self

Lys Differential band size

PCR indicates heterozygote
e

When progeny have been generated, genotype the

parental hermaphrodite to verify it was a cross
progeny by PCR
Step 3: Transfer progeny from plates that had
heterozygous parents to 96-well plates

Pare Parental Recombina Parental

nt Genotype nt Genotype

? ?
? Progeny genotypes are ?
unknown
? ? ?
?
Step 3: Transfer progeny from plates that had
heterozygous parents to 96-well plates

Pare Parental Recombina Parental

nt Genotype nt Genotype

Plates
include:
lysate
K-medium
Kanamycin?
 Step 4: Allow progeny to self and starve in
wells, then genotype each well
Plates
include:
lysate
K-medium
Kanamycin?

Pare Parental Recombina Parental

nt Genotype nt Genotype

Genotyping results for different

progeny
 Step 5: Identify recombinant
individuals
Plates
include:
lysate
K-medium
Kanamycin?

Pare Parental Recombina Parental

nt Genotype nt Genotype

Genotyping results for different

progeny
Step 6: Transfer recombinant progeny to agar plates
and allow to recover
Recombina
nt
 Step 7: Once recovered, transfer single worms to
individual plates to have single parental genotype
Recombina
nt
Step 8: Identify homozygous recombinant
progeny
Step 8: Identify homozygous recombinant
progeny
NILs recapitulate the QTL
phenotype
 NILs recapitulate the QTL
phenotype

mig-6 :: 7.83Mb lurp-4 :: 13.09Mb

(0.91cM) (4.87cM)
3.96
cM
2

0 7 o o

1 8 E E 1
 3.96
cM
We expect four recombinants in this interval for every 100
progeny

Setup 8 plates (768 progeny)

Expect ~ 31 recombinants within
interval
Arsenic Fraction
Adult
 Arsenic Fraction
Adult

frh-1 :: 5.9Mb (- sgn-1 :: 8.8Mb

0.85cM) (.9cM)
1.75
cM
2

0 6 o o

1 7 E E 1
 1.75
cM
We expect four recombinants in this interval for every 100
progeny

Setup 12 plates (1152 progeny)

Expect ~ 20 recombinants within
interval